Fungal Genet Biol, 1998 Jun-Jul, 24(1-2), 178 - 206 Dynamics of cell wall formation in fission yeast, Schizosaccharomyces pombe; Osumi M et al.; Studies on the dynamics of surface and intracellular structures during cell wall formation from the reverting protoplast of Schizosaccharomyces pombe were reviewed, and the correlation between cell wall formation and actin cytoskeleton, which is the most important conductor of the mechanism, is described in this paper . A close spatial and temporal relationship between actin cytoskeleton and cell wall formation was found by using wild type and actin point-mutant cps8 of S . pombe . Concomitant with the cell wall formation, dynamic behavior of the intracellular secretion machinery, especially the Golgi apparatus and secretory vesicles, was analyzed by three-dimensional reconstruction of 40 to 80 serial sections at five reverting stages . Total reverting protoplast volume increased by 3.8 and 4.3 times at 3 and 5 h, respectively, and the volume of the Golgi apparatus in the corresponding stages increased 2.3- and 2 . 5-fold over the same periods . The number of secretory vesicles also markedly increased by 3.4 and 5.8 times over that of the corresponding reverting protoplasts . Actin point-mutant cps8 cells have abnormal structure in the cell wall and septum, and the distribution pattern of the actin cytoskeleton during the reversion process was different from wild-type protoplasts . The profiles of actin showed one or two thick cables and patches in the cytoplasm which remained throughout reversion . The development of crosslinkage of the glucan fibrils which are beta-1,3-glucan in nature on the reverting protoplast surface was defective; the glucan networks consisted of thin, rope-shaped fibrils up to 30 nm in width which formed a ribbon-shape 200 nm wide in wild-type reverting protoplasts . The intrafibrillar space is not filled with amorphous particles of alpha-galactomannan in nature . The secretion machinery was seen to have a similar profile as the wild type . The above results suggest that actin cytoskeleton may control secretion of beta-1,6-glucan and other cell wall substances such as alpha-glucan and alpha-galactomannan rather than beta-1,3-glucan . Study of the role of actin cytoskeleton in the cell wall formation is contributing to the development of antifungal agents together with basic cell biology . Curr Biol, 1998 Sep 10, 8(18), 1023 - 6 A new large proteolytic complex distinct from the proteasome is present in the cytosol of fission yeast; Osmulski PA et al.; One eukaryotic proteolytic complex--the proteasome--is classed as the major nonlysosomal protease, by its known and suspected functions, its size and its complexity . It seems improbable that other enzymes may be capable of substituting, even partially, for the potent proteasome, as this complex has a vital role in many cellular processes . Nevertheless, it is possible to adapt cultured EL-4 mouse lymphoma cells to survive in the presence of a specific inhibitor of the proteasome . The inhibition of the proteasome in these adapted EL-4 cells is accompanied by a dramatic increase in the activity of a new, as yet uncharacterized, large proteolytic complex . Here, we have presented evidence that a similar proteolytic activity is constitutively present in fission yeast, Schizosaccharomyces pombe, and that the yeast and mouse enzymes share basic physicochemical properties . We have shown that the S . pombe protease is found in two stable oligomeric forms, both of which are peptidases, although only the larger form acts as a proteinase . The relative amounts of the large and the small forms of the protease in the complex depended on the growth phase of the yeast culture and affected enzyme activity, suggesting that the activity of the enzyme is regulated by its oligomerization status . We refer to the new proteolytic complex as the 'multicorn' to indicate its analogy to the archaebacterial tricorn protease. J Cell Sci, 1998 Oct, 111 ( Pt 20), 3101 - 8 Cdc18p can block mitosis by two independent mechanisms; Greenwood E et al.; The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed . The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis . The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle . We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p . The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis . The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes . Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner. Mol Gen Genet, 1998 Jul, 259(1), 123 - 9 Mapping of Rpb3 and Rpb5 contact sites on two large subunits, Rpb1 and Rpb2, of the RNA polymerase II from fission yeast; Miyao T et al.; {Rpb1 and Rpb2} Mapping of the contact sites on two large subunits of the fission yeast Schizosaccharomyces pombe RNA polymerase II with two small subunits, Rpb3 and Rpb5, was carried out using the two-hybrid screening system in the budding yeast Saccharomyces cerevisiae . Rpb5 was found to interact with any fragment of Rpb1 that contained the region H, which is conserved among the subunit 1 homologues of all RNA polymerases, including the beta' subunit of prokaryotic RNA polymerases . In agreement with the fact that Rpb5 is shared among all three forms of eukaryotic RNA polymerases, the region H of RNA polymerase I subunit 1 (Rpa190) was also found to interact with Rpb5 . On the other hand, two-hybrid screening of Rpb2 fragments from RNA polymerase II indicated the presence of an Rpb3 contact site in the region H which is conserved among the subunit 2 homologues of all RNA polymerases, including the beta subunit of prokaryotic RNA polymerases . Possible functions of the regions H in the subunits 1 and 2 are discussed. J Biol Chem, 1998 Sep 25, 273(39), 25098 - 105 Characterization of the recombinant MutY homolog, an adenine DNA glycosylase, from yeast Schizosaccharomyces pombe; Lu AL et al.; The mutY homolog (SpMYH) gene from a cDNA library of Schizosaccharomyces pombe encodes a protein of 461 amino acids that displays 28 and 31% identity to Escherichia coli MutY and human MutY homolog (MYH), respectively . Expressed SpMYH is able to complement an E . coli mutY mutant to reduce the mutation rate . Similar to E . coli MutY protein, purified recombinant SpMYH expressed in E . coli has adenine DNA glycosylase and apurinic/apyrimidinic lyase activities on A/G- and A/7,8-dihydro-8-oxoguanine (8-oxoG)-containing DNA . However, both enzymes have different salt requirements and slightly different substrate specificities . SpMYH has greater glycosylase activity on 2-aminopurine/G and A/2-aminopurine but weaker activity on A/C than E . coli MutY . Both enzymes also have different substrate binding affinity and catalytic parameters . Although SpMYH has great affinity to A/8-oxoG-containing DNA as MutY, the binding affinity to A/G-containing DNA is substantially lower for SpMYH than MutY . SpMYH has similar reactivity to both A/G- and A/8-oxoG-containing DNA; however, MutY cleaves A/G-containing DNA about 3-fold more efficiently than it does A/8-oxoG-containing DNA . Thus, SpMYH is the functional eukaryotic MutY homolog responsible for reduction of 8-oxoG mutational effect. Hum Mol Genet, 1998 Oct, 7(11), 1703 - 12 Characterization of the myotubularin dual specificity phosphatase gene family from yeast to human; Laporte J et al.; X-linked myotubular myopathy (XLMTM) is a severe congenital muscle disorder due to mutations in the MTM1 gene . The corresponding protein, myotubularin, contains the consensus active site of tyrosine phosphatases (PTP) but otherwise shows no homology to other phosphatases . Myotubularin is able to hydrolyze a synthetic analogue of tyrosine phosphate, in a reaction inhibited by orthovanadate, and was recently shown to act on both phosphotyrosine and phosphoserine . This gene is conserved down to yeast and strong homologies were found with human ESTs, thus defining a new dual specificity phosphatase (DSP) family . We report the presence of novel members of the MTM gene family in Schizosaccharomyces pombe, Caenorhabditis elegans, zebrafish, Drosophila, mouse and man . This represents the largest family of DSPs described to date . Eight MTM-related genes were found in the human genome and we determined the chromosomal localization and expression pattern for most of them . A subclass of the myotubularin homologues lacks a functional PTP active site . Missense mutations found in XLMTM patients affect residues conserved in a Drosophila homologue . Comparison of the various genes allowed construction of a phylogenetic tree and reveals conserved residues which may be essential for function . These genes may be good candidates for other genetic diseases. Genes Cells, 1998 Jun, 3(6), 347 - 55 Defect in cytokinesis of fission yeast induced by mutation in the WD40 repeat motif of a TFIID subunit; Yamamoto T et al.; BACKGROUND: TBP-associated factors contain a variety of structural motifs and their related in vivo significance has remained unclear . We have attempted to identify specific biological phenomena linked to a particular domain of a TAF by analysing domain-exchanged chimeric mutants between Schizosaccharomyces pombe (Sp) and Saccharomyces cerevisiae (Sc) counterparts . RESULTS: Contrary to the case of TBP, Sp TAF containing the WD40 repeat cannot be exchanged for its Sc counterpart, despite their highly conserved primary structures . This 'species-specific' function locates in the N-terminal region . The C-terminal region, largely consisting of the WD40 repeat, is exchangeable for the corresponding region of its Sc counterpart . Growth of the strain harbouring this C-terminal chimeric mutant is temperature-sensitive . The chimeric gene product did not disappear at a restrictive temperature, a finding which strongly suggests that the growth defect is caused by an aberration in the interactions through the WD40 repeat structural motif . With temperature elevation, the chimeric mutants underwent drastic morphological changes due to a defect in cytokinesis . CONCLUSIONS: The WD40 repeat of TAF is primarily involved in reactions which might regulate cytokinesis in Sp. Yeast, 1998 Aug, 14(11), 1051 - 9 The uracil permease of Schizosaccharomyces pombe: a representative of a family of 10 transmembrane helix transporter proteins of yeasts; de Montigny J et al.; The uracil permease gene of Schizosaccharomyces pombe was cloned and sequenced . The deduced protein sequence shares strong similarities with five open reading frames from Saccharomyces cerevisiae, namely the uracil permease encoded by the FUR4 gene, the allantoin permease encoded by DAL4, a putative uridine permease (YBL042C) and two unknown ORFs YOR071c and YLR237w . A topological model retaining ten transmembrane helices, based on predictions and on experimental data established for the uracil permease of S . cerevisiae by Galan and coworkers (1996), is discussed for the four closest proteins of this family of transporters . The sequence of the uracil permease gene of S . pombe has been deposited in the EMBL data bank under Accession Number X98696. Biochem J, 1998 Sep 15, 334 ( Pt 3), 609 - 16 Human and mouse Gpi1p homologues restore glycosylphosphatidylinositol membrane anchor biosynthesis in yeast mutants; Tiede A et al.; Glycosylphosphatidylinositol (GPI) represents an important anchoring molecule for cell surface proteins . The first step in its synthesis is the transfer of N-acetylglucosamine (GlcNAc) from UDP to phosphatidylinositol (PI) . The products of three mammalian genes, PIG-A, PIG-C and PIG-H, have previously been shown to be involved in the putative enzymic complex . Here we report the cloning of human and mouse cDNAs encoding a fourth participant in the GlcNAc transfer reaction which are homologues of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Gpi1 proteins . To provide evidence for their function, these proteins were expressed in GPI1-disrupted yeast strains . In Sacch . cerevisiae, where GPI1 disruption results in a temperature-sensitive phenotype and abolishes in vitro GlcNAc-PI synthesis, restoration of growth could be demonstrated in a temperature-dependent manner . In addition, in vitro GlcNAc-PI synthetic activity was again detectable . In Schiz . pombe, gpi1+ disruption is lethal . Using random spore analysis, we were able to show that the mammalian GPI1 homologues can rescue haploids harbouring the lethal gpi1+::his7+ allele . Our data demonstrate that the genes identified are indeed involved in the first step of GPI biosynthesis, and allow conclusions about a specific function for Gpi1p in stabilizing the enzymic complex . The finding that, despite a low degree of identity, the mammalian Gpi1 proteins are able to participate in the yeast GlcNAc-PI synthetic machinery as heterologous components further demonstrates that GPI biosynthesis has been highly conserved throughout evolution. Biochim Biophys Acta, 1998 Aug 24, 1381(3), 271 - 8 Trehalose-6P synthase is essential for trehalase activation triggered by glucose, nitrogen source or heat shock, but not by osmostress, in Schizosaccharomyces pombe; Cansado J et al.; Cells of Schizosaccharomyces pombe disrupted in the tps1+ gene, which encodes trehalose-6P synthase, were unable to increase trehalase activity in response to the addition of glucose or nitrogen source . Moreover, in contrast to normal cells, Deltatps1 cells did not increase trehalase activity by heat shock . Overexpression of tps1+ in cells devoid of trehalose-6P synthase restored the ability to increase trehalase after addition of nutrients or by heat shock . In glucose-repressed cells, which are normally refractory to the activation of trehalase by glucose, overexpression of tps1+ enabled the cells to increase trehalase activity upon addition of the sugar . Northern hybridisations were used to determine the level of mRNA for trehalase in normal and Deltatps1 cells . Transcription for trehalase was not significantly altered upon addition of glucose or nitrogen source, but increased markedly in heat-shocked cells even though trehalase activity remained unchanged in Deltatps1 cells . These findings provide evidence for a role of trehalose-6P synthase in the signalling pathway causing post-transcriptional activation of neutral trehalase induced by nutrients or heat shock . However, trehalase increased in Deltatps1 cells under hypertonic conditions suggesting the existence in Schiz . pombe of a distinct regulatory mechanism for enhancement of trehalase, specifically triggered by osmostress . Copyright Elsevier Science B.V. J Biol Chem, 1998 Sep 11, 273(37), 23938 - 45 The Pad1+ gene encodes a subunit of the 26 S proteasome in fission yeast; Penney M et al.; We have isolated a fission yeast mutant, mts5-1, in a screen for mutations that confer both methyl 2-benzimidazolecarbamate resistance (MBCR) and temperature sensitivity (ts) on Schizosaccharomyces pombe . This screen has previously isolated mutations in the 26 S proteasome subunits Mts2, Mts3, and Mts4 . We show that the mutation in the mts5-1 strain occurs in the pad1(+) gene . pad1(+) was originally isolated on a multicopy plasmid that was capable of conferring staurosporine resistance on a wild type strain . mts5-1/pad1-1 has a similar phenotype to 26 S proteasome mutants previously isolated in the same screen and we show that Pad1 interacts genetically with two of these subunits, Mts3 and Mts4 . In this study we describe the identification of Pad1 as a subunit of the 26 S proteasome in fission yeast. Nucleic Acids Res, 1998 Sep 15, 26(18), 4222 - 9 The fission yeast chromo domain encoding gene chp1(+) is required for chromosome segregation and shows a genetic interaction with alpha-tubulin; Doe CL et al.; In eukaryotes, the segregation of chromosomes is co-ordinated by the centromere and must proceed accurately if aneuploidy and cell death are to be avoided . The fission yeast centromere is complex, containing highly repetitive regions of DNA showing the characteristics of heterochromatin . Two proteins, Swi6p and Clr4p, that are associated with the fission yeast centromere also contain a chromo (chromatin organisation modifier) domain and are required for centromere function . We have analysed a novel fission yeast gene encoding a putative chromo domain called chp 1(+) (chromo domain protein in Schizosaccharomyces p ombe ) . In the absence of Chp1p protein, cells are viable but show chromosome segregation defects such as lagging chromosomes on the spindle during anaphase and high rates of minichromosome loss, phenotypes which are also displayed by swi 6 and clr 4 . A fusion protein between green fluorescent protein (GFP) and Chp1p, like Swi6p, is localized to discrete sites within the nucleus . In contrast to Swi6p and Clr4p, Chp1p is not required to repress silent mating-type genes . We demonstrate a genetic interaction between chp 1(+) and alpha-tubulin ( nda 2(+)) and between swi 6(+) and beta-tubulin ( nda 3(+)) . Chp1p and Swi6p proteins may be components of the kinetochore which captures and stabilizes the microtubules of the spindle. Microbiology, 1998 Aug, 144 ( Pt 8), 2331 - 44 Cytochalasin D interferes with contractile actin ring and septum formation in Schizosaccharomyces japonicus var . versatilis; Gabriel M et al.; The cells of Schizosaccharomyces japonicus var . versatilis responded to the presence of cytochalasin D (CD), an inhibitor of actin polymerization, by the disappearance of contractile actin rings (ARs) that had already formed and by inhibition of new ring formation . Actin cables disappeared . Actin patches remained preserved and became co-localized with regions of actual cell wall formation (at cell poles and at the site of septum development) . Removal of the AR arrested formation of the primary septum and led to the production of aberrant septum protrusions in that region . Nuclear division was accomplished in the presence of CD but new ARs were not produced . The wall (septum) material was deposited in the form of a wide band at the inner surface of the lateral cell wall in the cell centre . This layer showed a thin fibrillar structure . The removal of CD resulted in rapid formation of new ARs in the equatorial region of the cells . This implies that the signal for AR localization was not abolished either by CD effects or by removal of an AR already formed . Some of the newly developed ARs showed atypical localization and orientation . In addition, redundant, subcortically situated actin bundles were produced . The removal of CD was quickly followed by the development of primary septa co-localized with ARs . Wall protrusions occurred co-localized with the redundant actin bundles . If these were completed in a circle, redundant septa developed . The AR is a mechanism which, in time and space, triggers cytokinesis by building a septum sequentially dependent on the AR . Aberrant septa were not capable of separating daughter cells . However, non-separated daughter cells subsequently gave rise to normal cells. Yeast, 1998 Jul, 14(10), 953 - 61 Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae; Longtine MS et al.; An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes . This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest . We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications . Using as selectable marker the S . cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging {with GFP(S65T), GST, or the 3HA or 13Myc epitope}, and partial N- or C-terminal deletions (with or without concomitant protein tagging) . Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations . Thus, these plasmids should further facilitate the rapid analysis of gene function in S . cerevisiae. Yeast, 1998 Jul, 14(10), 943 - 51 Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe; Bahler J et al.; We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe . Using this approach and the S . pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63% . We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S . pombe . The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging . Nine genes were manipulated using these kanMX6 constructs as templates for PCR . The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome . Transformants were screened for homologous integration by PCR . In most cases, the efficiency of homologous integration was > or = 50%, and the lowest efficiency encountered was 17% . The methodology and constructs described here should greatly facilitate analysis of gene function in S . pombe. Antonie Van Leeuwenhoek, 1998 Feb, 73(2), 189 - 94 Deleterious effects of androstenedione on growth and cell morphology of Schizosaccharomyces pombe; Dlugonski J et al.; Androgens (androstenedione and testosterone) belong to the most important compounds in human steroidogenesis . The 17 beta-hydroxysteroid dehydrogenase responsible for interconversion of the oxygenic group on C-17 of androgens ring is involved in steroid hormone synthesis . The fission yeast Schizosaccharomyces pombe 972 h- was found to contain constitutive 17 beta-hydroxysteroid dehydrogenase that was able to reduce androstenedione to testosterone and oxidize testosterone to androstenedione . The reductive pathway was found to be predominant while the oxidative one was carried out with much lower activity . Exogenous androstenedione, contrary to testosterone, inhibited S . pombe growth and stimulated the formation of aberrant swollen cells with slighter cell wall sensitivity to the action of the lytic enzyme Novozym . It is postulated that the 17 beta-hydroxysteroid dehydrogenase prevents the deleterious effects of androstenedione on the morphology and growth of the yeast's cells by androstedione reduction to testosterone. Biochem J, 1998 Sep 1, 334 ( Pt 2), 377 - 86 Isolation and characterization of a processive DNA helicase from the fission yeast Schizosaccharomyces pombe that translocates in a 5'-to-3' direction; Lee C et al.; We report here the isolation and characterization of a novel DNA helicase from extracts of the fission yeast Schizosaccharomyces pombe . The enzyme, called DNA helicase II, also contains an intrinsic DNA-dependent ATPase activity . Both the helicase and ATPase activities co-purified with a 63 kDa polypeptide on an SDS/polyacrylamide gel . The protein has a sedimentation coefficient of 4.8 S and a Stokes radius of 36 A (3.6 nm); from these data the native molecular mass was calculated to be 65 kDa . The enzyme translocates in a 5'-to-3' direction with respect to the substrate strand to which it is bound . Unwinding reactions carried out in the presence of increasing enzyme showed a sigmoidal curve, suggesting either co-operative interactions between monomers or multimerization of DNA helicase II in the presence of single-stranded DNA and/or ATP . This enzyme favoured adenosine nucleotides (ATP and dATP) as its energy source, but utilized to limited extents GTP, CTP, dGTP and dCTP . Non-hydrolysable ATP analogues did not support helicase activity . Kinetic analyses showed that the unwinding reaction was rapid, being complete after 50-100 s of incubation . Addition of unlabelled substrates to the helicase reaction after preincubation of the enzyme with substrate did not significantly diminish unwinding . The ATPase activity of DNA helicase II increased proportionally with increasing lengths of single-stranded DNA cofactor . In the presence of circular DNA, ATP hydrolysis continued to increase up to the longest time tested (3 h), whereas it ceased to increase after 5-10 min in the presence of shorter oligonucleotides . The initial rate of ATP hydrolysis during the first 5 min of incubation time was not affected by DNA species used . These data indicate that the enzyme does not dissociate from the single-stranded DNA once it is bound and is therefore highly processive. Genes Dev, 1998 Aug 15, 12(16), 2560 - 73 Human and mouse homologs of Schizosaccharomyces pombe rad1(+) and Saccharomyces cerevisiae RAD17: linkage to checkpoint control and mammalian meiosis; Freire R et al.; Preventing or delaying progress through the cell cycle in response to DNA damage is crucial for eukaryotic cells to allow the damage to be repaired and not incorporated irrevocably into daughter cells . Several genes involved in this process have been discovered in fission and budding yeast . Here, we report the identification of human and mouse homologs of the Schizosaccharomyces pombe DNA damage checkpoint control gene rad1(+) and its Saccharomyces cerevisiae homolog RAD17 . The human gene HRAD1 is located on chromosome 5p13 and is most homologous to S . pombe rad1(+) . This gene encodes a 382-amino-acid residue protein that is localized mainly in the nucleus and is expressed at high levels in proliferative tissues . This human gene significantly complements the sensitivity to UV light of a S . pombe strain mutated in rad1(+) . Moreover, HRAD1 complements the checkpoint control defect of this strain after UV exposure . In addition to functioning in DNA repair checkpoints, S . cerevisiae RAD17 plays a role during meiosis to prevent progress through prophase I when recombination is interrupted . Consistent with a similar role in mammals, Rad1 protein is abundant in testis, and is associated with both synapsed and unsynapsed chromosomes during meiotic prophase I of spermatogenesis, with a staining pattern distinct from that of the recombination proteins Rad51 and Dmc1 . Together, these data imply an important role for hRad1 both in the mitotic DNA damage checkpoint and in meiotic checkpoint mechanisms, and suggest that these events are highly conserved from yeast to humans. Gene, 1998 Jul 30, 215(2), 319 - 28 Molecular cloning of gaf1, a Schizosaccharomyces pombe GATA factor, which can function as a transcriptional activator; Hoe KL et al.; As a first step to elucidate the functions of Schizosaccharomyces pombe (S . pombe) GATA factors, we have isolated the gaf1+ gene (GATA-factor like gene) in S . pombe . The predicted amino acid (aa) sequence of Gaf1 reveals a single zinc finger domain typical of fungal GATA factors, and the zinc finger exhibits 60% aa identity to that of human GATA-1 . The open reading frame of Gaf1 predicts a protein of Mr 32 kDa consisting of 290 intronless amino acids . Disruption of this gene has no effect on cell viability and growth rate . The GST-Gaf1 fusion protein binds specifically to GATA motifs of its own promoter as well as DAL7 UAS, a canonical GATA motif of Saccharomyces cerevisiae (S . cerevisiae) The specific DNA-binding activity resides within the N-terminal half of Gaf1 (Gaf1N; aa 1-120) containing the zinc finger, whereas the C-terminal half (Gaf1C; aa 121-290) contains transactivation sequences that induce the expression of the lacZ reporter when fused to the GAL4 DNA binding domain . These results demonstrate that Gaf1 may function as a transcriptional activator consisting of DNA-binding and transactivation domains. Biochim Biophys Acta, 1998 Jul 30, 1399(1), 67 - 72 A Schizosaccharomyces pombe gene encoding a novel polypeptide with a predicted alpha-helical rod structure found in the myosin and intermediate-filament families of proteins; Jannatipour M et al.; We have identified a Schizosaccharomyces pombe gene encoding a 461 amino acid polypeptide containing a predicted alpha-helical rod domain found in filamentous proteins . This gene, here designated noc1, is located immediately upstream from cnx1, the gene encoding the S . pombe homologue of mammalian calnexin, an endoplasmic reticulum chaperone {M . Jannatipour, L.A . Rokeach, J . Biol . Chem . 270 (1995) 4845-4853.} . Transcription of noc1 is divergent from that of cnx1 . Northern blot analysis identified a single mRNA of approx . 2 kb whose expression was increased by heat shock and growth in the presence of beta-mercaptoethanol and 2-deoxyglucose. Mol Cell Biol, 1998 Sep, 18(9), 5511 - 22 A novel function of the DNA repair gene rhp6 in mating-type silencing by chromatin remodeling in fission yeast; Singh J et al.; Recent studies have indicated that the DNA replication machinery is coupled to silencing of mating-type loci in the budding yeast Saccharomyces cerevisiae, and a similar silencing mechanism may operate in the distantly related yeast Schizosaccharomyces pombe . Regarding gene regulation, an important function of DNA replication may be in coupling of faithful chromatin assembly to reestablishment of the parental states of gene expression in daughter cells . We have been interested in isolating mutants that are defective in this hypothesized coupling . An S . pombe mutant fortuitously isolated from a screen for temperature-sensitive growth and silencing phenotype exhibited a novel defect in silencing that was dependent on the switching competence of the mating-type loci, a property that differentiates this mutant from other silencing mutants of S . pombe as well as of S . cerevisiae . This unique mutant phenotype defined a locus which we named sng1 (for silencing not governed) . Chromatin analysis revealed a switching-dependent unfolding of the donor loci mat2P and mat3M in the sng1(-) mutant, as indicated by increased accessibility to the in vivo-expressed Escherichia coli dam methylase . Unexpectedly, cloning and sequencing identified the gene as the previously isolated DNA repair gene rhp6 . RAD6, an rhp6 homolog in S . cerevisiae, is required for postreplication DNA repair and ubiquitination of histones H2A and H2B . This study implicates the Rad6/rhp6 protein in gene regulation and, more importantly, suggests that a transient window of opportunity exists to ensure the remodeling of chromatin structure during chromosome replication and recombination . We propose that the effects of the sng1(-)/rhp6(-) mutation on silencing are indirect consequences of changes in chromatin structure. Mol Cell Biol, 1998 Sep, 18(9), 5239 - 46 The role of fnx1, a fission yeast multidrug resistance protein, in the transition of cells to a quiescent G0 state; Dimitrov K et al.; Most microorganisms live in conditions of nutrient limitation in their natural habitats . When exposed to these conditions they respond with physiological and morphological changes that enable them to survive . To obtain insights into the molecular mechanisms of this response a systematic genetic screen was performed to identify genes that when overexpressed can induce a starvation-like response in the yeast species Schizosaccharomyces pombe . One gene that meets these criteria, fnx1(+), induces, transcriptionally correlates with, and is required for the entry into the quiescent G0 state that is normally induced by nitrogen starvation . fnx1(+) encodes a protein with sequence similarity to the proton-driven plasma membrane transporters from the multidrug resistance group of the major facilitator superfamily of proteins . We propose that fnx1(+) plays a role in the entry into G0, possibly by facilitating the release of a signaling substance into the environment as a means of cell-to-cell communication. Biochemistry, 1998 Aug 18, 37(33), 11599 - 604 Expression, purification, and characterization of ultraviolet DNA endonuclease from Schizosaccharomyces pombe; Kaur B et al.; Ultraviolet damage endonuclease (UVDE) is a 68.7 kDa DNA repair enzyme of Schizosaccharomyces pombe that recognizes cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs) . UVDE is thought to initiate the first step in an alternative excision repair pathway for removal of UV light-induced DNA damage . We have overexpressed Delta228-UVDE, an active truncated form of UVDE, and have purified this protein to apparent homogeneity . We have characterized purified Delta228-UVDE with respect to its physical properties, divalent cation requirements, and kinetic parameters on oligodeoxynucleotide substrates containing a single CPD . DNA strand cleavage analysis indicates that both full-length UVDE and Delta228-UVDE incise the CPD-containing strand immediately 5' to the lesion . These results provide further insight into the UVDE-mediated alternative excision repair pathway. Nucleic Acids Res, 1998 Sep 1, 26(17), 3955 - 60 Schizosaccharomyces pombe Mcm3p, an essential nuclear protein, associates tightly with Nda4p (Mcm5p); Sherman DA et al.; MCM proteins are required for the proper regulation of DNA replication . There are six MCM proteins in all eukaryotes which interact to form a large complex . We report the cloning of fission yeast mcm3 + . mcm3 + is essential and spores carrying a Delta mcm3 disruption arrest with an apparently replicated DNA content . The protein is found constitutively in the nucleus and levels remain constant throughout the cell cycle . Mcm3p binds particularly tightly to Nda4p (Mcm5p), but is loosely associated with the other Schizosaccharomyces pombe MCM proteins . Thus, Mcm3p is a peripheral MCM subunit. J Mol Biol, 1998 Jul 31, 280(5), 811 - 21 Site-specific insertion of a SINE-like element, Cp1, into centromeric tandem repeats from Chironomus pallidivittatus; Liao C et al.; A SINE-like dispersed element, Cp1, from the dipteran Chironomus pallidivittatus was found to show site-specific insertion into two different centromeric tandem repeats . The insertions result in identical target site duplications of nine base-pairs . In contrast, extracentromeric Cp1 elements, which are polymorphic and degenerate, are previously known to be surrounded by different target site duplications . The intracentromeric Cp1 is uniform in structure and contains a single pol III unit, upstream of which 87 bp arms of a palindrome surround a 103 bp unique sequence . The numbers of Cp1 elements per centromere were determined in microdissected material and were found to be in the range of five to ten units per centromere . The well-defined insertion properties, correlated to chromosomal localization, suggest that Cp1 is likely to be a component of importance for the centromere . Similarities of Cp1 and its parts to functionally identified centromeres in Saccharomyces cerevisiae and Schizosaccharomyces pombe are discussed . RNA, 1998 Aug, 4(8), 958 - 72 The stretch of C-terminal acidic amino acids of translational release factor eRF1 is a primary binding site for eRF3 of fission yeast; Ito K et al.; Translation termination in eukaryotes requires a codon-specific (class-I) release factor, eRF1, and a GTP/GDP-dependent (class-II) release factor, eRF3 . The model of "molecular mimicry between release factors and tRNA" predicts that eRF1 mimics tRNA to read the stop codon and that eRF3 mimics elongation factor EF-Tu to bring eRF1 to the A site of the ribosome for termination of protein synthesis . In this study, we set up three systems, in vitro affinity binding, a yeast two-hybrid system, and in vitro competition assay, to determine the eRF3-binding site of eRF1 using the fission yeast Schizosaccharomyces pombe proteins and creating systematic deletions in eRF1 . The in vitro affinity binding experiments demonstrated that the predicted tRNA-mimicry truncation of eRF1 (Sup45) forms a stable complex with eRF3 (Sup35) . All three test systems revealed that the most critical binding site is located at the C-terminal region of eRF1, which is conserved among eukaryotic eRF1s and rich in acidic amino acids . To our surprise, however, the C-terminal deletion eRF1 seems to be sufficient for cell viability in spite of the severe defect in eRF3 binding when expressed in a temperature-sensitive sup45 mutant of the budding yeast, Saccharomyces cerevisiae . These results cannot be accounted for by the simple "eRF3-EF-Tu mimicry" model, but may provide new insight into the eRF3 function for translation termination in eukaryotes. Trends Cell Biol, 1998 Apr, 8(4), 163 - 7 Discovering the poles in yeast; Mata J et al.; How cells generate and orientate polarized growth is of fundamental importance to understanding cell morphogenesis . The budding yeast Saccharomyces cerevisiae and the distantly related fission yeast Schizosaccharomyces pombe have both been used for genetic analysis of cell morphogenesis . Generation and maintenance of their cell shape require the formation of polarized growth sites and the correct localization of these growth sites on the cell surface with respect to other cellular structures . In this review, the authors discuss and compare the mechanisms used by the two yeasts to achieve polarized growth. Trends Cell Biol, 1998 Apr, 8(4), 144 - 9 Fission yeast cut mutations revisited: control of anaphase; Yanagida M; Studies of anaphase are approaching a golden age . Several different disciplines have contributed immensely to advances in our understanding of cell-cycle control and chromosome and spindle dynamics during mitosis . This article describes control of anaphase based on results obtained from Schizosaccharomyces pombe cut (cell untimely torn) mutants . These temperature-sensitive mutants were isolated by selection for uncoordinated mitosis with aberrant sister-chromatid separation and post-anaphase events . Characterization of some of the cut gene products has led to identification of novel molecular events related to chromosome condensation, sister-chromatid separation, anaphase-promoting proteolysis, fatty-acid metabolism, and cell-cycle arrest induced by stress or a replication block. Mol Biol Cell, 1998 Aug, 9(8), 2325 - 35 The fission yeast mitotic regulator win1+ encodes an MAP kinase kinase kinase that phosphorylates and activates Wis1 MAP kinase kinase in response to high osmolarity; Samejima I et al.; The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle . Although the defect is suppressed by wis1+ and wis4+, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known . We show here that win1+ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1 . Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions . Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations . The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain . We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes. Mol Biol Cell, 1998 Aug, 9(8), 2231 - 47 The Saccharomyces cerevisiae prenylcysteine carboxyl methyltransferase Ste14p is in the endoplasmic reticulum membrane; Romano JD et al.; Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation . We demonstrated previously that the STE14 gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast . In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay . We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast . In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins . Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face . Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi . We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S . cerevisiae and have shown that mam4p complements a Deltaste14 mutant . This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues. Mol Biol Cell, 1998 Aug, 9(8), 2011 - 23 The role of the Schizosaccharomyces pombe gar2 protein in nucleolar structure and function depends on the concerted action of its highly charged N terminus and its RNA-binding domains; Sicard H et al.; Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe . We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure . Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain . These drastic functional defects correlate with striking nucleolar hypertrophy . Point mutations in the conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA-protein interactions are sufficient to induce severe nucleolar modifications but only in the presence of the N-terminal domain of the protein . Gar2 and its mutants also distribute differently in glycerol gradients: gar2 lacking its RBDs is found either free or assembled into significantly larger complexes than the wild-type protein . We propose that gar2 helps the assembly on rRNA of factors necessary for 40S subunit synthesis by providing a physical link between them . These factors may be recruited by the N-terminal domain of gar2 and may not be released if interaction of gar2 with rRNA is impaired. Protein Expr Purif, 1998 Aug, 13(3), 403 - 13 Cloning of the fatty acid synthetase beta subunit from fission yeast, coexpression with the alpha subunit, and purification of the intact multifunctional enzyme complex; Niwa H et al.; We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe) fas1+ gene, which encodes the fatty acid synthetase (FAS) beta subunit, by applying a PCR technique to conserved regions in the beta subunit of the alpha6beta6 types of FAS among different organisms . The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (Mr = 230,616), exhibits the 48.1% identity with the beta subunit from the budding yeast (Saccharomyces cerevisiae) . This subunit, with five different catalytic activities, bears four distinct domains, while the alpha subunit, the sequence of which was previously reported by Saitoh et al . (S . Saitoh et al., 1996, J . Cell Biol . 134, 949-961), carries three domains . We have developed a co-expression system of the FAS alpha and beta subunits by cotransformation of two expression vectors, containing the lsd1+/fas2+ gene and the fas1+ gene, into fission yeast cells . The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification . Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1-2.4 x 10(6), and one molecule of the FAS complex was found to contain approximately six FMN molecules . These results indicate that the FAS complex from S . pombe forms a heterododecameric alpha6beta6 structure . Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture . Genetics, 1998 Aug, 149(4), 1753 - 61 Molecular analysis of pcc1, a gene that leads to A-regulated sexual morphogenesis in Coprinus cinereus; Murata Y et al.; A homokaryotic strain (5337) in our culture stock of Coprinus cinereus produced fertile fruit bodies after prolonged culture . Microscopic examination revealed that hyphae dedifferentiated from the tissues of one of the fruit bodies, as well as all basidiospore derivatives from the fruit body, exhibited pseudoclamps, whereas vegetative hyphae of 5337, from which the fruit body developed, had no clamp connections . Genetic analysis showed that the formation of pseudoclamps results from a recessive mutation in a gene designated pcc1 (pseudoclamp connection formation), which is distinct from the A and B mating type genes . Cloning and sequencing of the pcc1 gene and cDNA identified an ORF of 1683 bp interrupted by one intron . Database searches revealed that pcc1 encodes an SRY-type HMG protein . The HMG box shared 44, 41, and 29% sequence identities (>80 amino acids) to those of FPR1 of Podospora anserina, MAT-Mc of Schizosaccharomyces pombe, and prf1 of Ustilago maydis, respectively . Northern analysis revealed that the level of pcc1 expression is higher in the dikaryon, in homokaryons in which the A and B mating type developmental sequences are individually activated, than in the homokaryon in which these sequences are not active . Sequencing of the pcc1-1 mutant allele revealed that the mutant carries a nonsense mutation at serine 211, a residue located between the HMG box and the C terminus . Based on these results, possible roles of the pcc1 gene in the sexual development of homobasidiomycetes are discussed. Genetics, 1998 Aug, 149(4), 1729 - 37 The Schizosaccharomyces pombe S-phase checkpoint differentiates between different types of DNA damage; Rhind N et al.; We have identified an S-phase DNA damage checkpoint in Schizosaccharomyces pombe . This checkpoint is dependent on Rad3, the S . pombe homolog of the mammalian ATM/ATR checkpoint proteins, and Cds1 . Cds1 had previously been believed to be involved only in the replication checkpoint . The requirement of Cds1 in the DNA damage checkpoint suggests that Cds1 may be a general target of S-phase checkpoints . Unlike other checkpoints, the S . pombe S-phase DNA damage checkpoint discriminates between different types of damage . UV-irradiation, which causes base modification that can be repaired during G1 and S-phase, invokes the checkpoint, while gamma-irradiation, which causes double-stranded breaks that cannot be repaired by a haploid cell if induced before replication, does not invoke the checkpoint . Because the same genes are required to respond to UV- and gamma-irradiation during G2, this discrimination may represent an active suppression of the gamma response during S-phase. EMBO J, 1998 Aug 3, 17(15), 4503 - 10 A site- and strand-specific DNA break confers asymmetric switching potential in fission yeast; Arcangioli B; Mating-type switching in the fission yeast Schizosaccharomyces pombe results in the transfer of genetic information from one of the two silent cassettes (mat2P or mat3M) to the transcriptionally active locus (mat1) . The switching pattern is programmed by an imprinting event which restricts mat1 gene conversion to only one of the two sister cells, leading to asymmetric cell division . Biochemical analysis indicated that the mat1 locus contains a fragile chromosomal site . Southern hybridization and primer extension experiments showed that the fragility consists of a single-strand break (SSB) . The nicked DNA is stable throughout the cell cycle . The features of the nick fulfil all the requirements for the 'epigenetic', site and strand-specific chromosome modification at the mat1 locus, providing strong evidence that an SSB can initiate mitotic and meiotic gene conversion during replication. Nucleic Acids Res, 1998 Aug 15, 26(16), 3762 - 8 Hex1: a new human Rad2 nuclease family member with homology to yeast exonuclease 1; Wilson DM 3rd et al.; Nucleolytic processing of chromosomal DNA is required in operations such as DNA repair, recombination and replication . We have identified a human gene, named HEX1 forhumanexonuclease 1, by searching the EST database for cDNAs that encode a homolog to the Saccharomyces cerevisiae EXO1 gene product . Based on its homology to this and other DNA repair proteins of the Rad2 family, most notably Schizosaccharomyces pombe exonuclease 1 (Exo1), Hex1 presumably functions as a nuclease in aspects of recombination or mismatch repair . Similar to the yeast proteins, recombinant Hex1 exhibits a 5'-->3' exonuclease activity . Northern blot analysis revealed that HEX1 expression is highest in fetal liver and adult bone marrow, suggesting that the encoded protein may operate prominently in processes specific to hemopoietic stem cell development . HEX1 gene equivalents were found in all vertebrates examined . The human gene includes 14 exons and 13 introns that span approximately 42 kb of genomic DNA and maps to the chromosomal position 1q42-43, a region lost in some cases of acute leukemia and in several solid tumors. Nucleic Acids Res, 1998 Aug 15, 26(16), 3645 - 50 Characterization of Schizosaccharomyces pombe Rad2 protein, a FEN-1 homolog; Alleva JL et al.; FEN-1 proteins are a family of nucleases essential for lagging strand DNA synthesis . A gene with sequence similarity to FEN-1 protein-encoding genes, rad2 +, has been identified in Schizosaccharomyces pombe . We report the overexpression, purification, and character-ization of the putative S.pombe FEN-1 homolog, Rad2p . A GST-Rad2p fusion protein was over-expressed in Saccharomyces cerevisiae and purified to near homogeneity by GST affinity chromatography . Although Rad2p had been previously classified as a putative FEN-1 protein based on amino acid homology, there has been no biochemical evidence demonstrating flap endonuclease activity . DNA cleavage analysis of several different oligodeoxynucleotide structuresindicates that GST-Rad2p possesses both 5'-flap endonuclease and 5'-->3' double-stranded DNA exo-nuclease activities . GST-Rad2p incises a 5'-flap and a 5'-pseudo-Y structure one base 3' of the branch point in the duplex region and also degrades double-stranded DNA . This is the first report on the biochemical characterization of S.pombe Rad2p . The potential roles of Rad2p in DNA excision repair and other nucleic acid reactions are discussed. Micron, 1998 Apr-Jun, 29(2-3), 207 - 33 The ultrastructure of yeast: cell wall structure and formation; Osumi M; Yeasts are unicellular eukaryotes, and are used widely as a model system in basic and applied fields of life science, medicine, and biotechnology . The ultrastructure of yeast cells was first studied in 1957 and the techniques used have advanced greatly in the 40 years since then; an overview of these methods is first presented in this review . The ultrastructure of budding and dimorphic yeast cells observed with a scanning electron microscope (SEM) and a transmission electron microscope (TEM) after thin sectioning and freeze-etching are then described, followed by discussion of the regeneration of the cell wall of Candida albicans protoplasts detected by cryosectioning . C . albicans protoplasts are regenerated to synthesize microfibrils on their surface . They are aggregated into thicker bundles which are intermeshed, forming a wide-meshed network of long fibrils . These microfibrillar structures are chains of beta-1,3-glucan which are broken down after treatment with beta-1,3-glucanase . Morphologically identical microfibrils are synthesized in vitro by a cell-free system in which the active cell membrane fraction as a source of beta-1,3-glucan synthetase and UDP glucose as the sole substrate are used . The diameter of an elemental fibril of beta-glucan is estimated to be 2.8 nm from the pattern of autocorrelation of the image obtained by computer processing . In contrast, in the presence of aculeacin A the formation of normal fibrillar nets or bundles is significantly inhibited, resulting in the occurrence of short fibrils . These electron microscopic data suggest that aculeacin A inhibits not only the synthesis of beta-1,3-glucan but the aggregation of microfibrils of this polysaccharide, allowing formation of the crystalline structure . On the basis of the cumulative data obtained from the electron microscopic studies, we are led to the assumption that de novo synthesized beta-glucan chains might initially form fine particles which are then transformed into thin fibrils with single to multiple strands which appear to be oriented parallel to each other so that they develop into fibrillar structures . This process of assembly of beta-glucan molecules leads to the development of a fibrous network within the regenerating Candida cell wall . Third, the mechanism of cell wall formation is shown by low-voltage (LV) SEM and TEM, using various techniques and computer graphics, of the regeneration system of Schizosaccharomyces pombe protoplasts: after 10 min of regeneration, the protoplasts begin to grow fibrillar substances of a beta-glucan nature, and a fibrillar network covers the surface of all protoplasts . The network is originally formed as fine particles on the protoplast surface and these are subsequently lengthened to microfibrils 2 nm thick . The microfibrils twist around each other and develop into 8 nm thick fibrils forming flat bundles 16 nm thick . Interfibrillar spaces are gradually filled with amorphous particles of an alpha-galactomannan nature and, finally, the complete cell wall is formed after 12 h . Treatment of reverting protoplasts with RuO4 provided clear TEM images of glucan fibrils with high electron density . The relationship between cell wall regeneration and intracellular organelles was examined by using serial thin sections stained with PATAg and computer-aided three-dimensional reconstruction . The secretory vesicles in a protoplast had increased markedly by 1.4, 3.4, and 5.8 times at 1.5, 3.0, and 5 h, respectively . Three-dimensional analysis indicates that Golgi apparatuses are located close together in the nucleus of the protoplast and are dispersed into the cytoplasm during the progress of cell wall formation. J Bacteriol, 1998 Aug, 180(15), 3992 - 6 Multicellular stalk-like structures in Saccharomyces cerevisiae; Engelberg D et al.; Stalk formation is a novel pattern of multicellular organization . Yeast cells which survive UV irradiation form colonies that grow vertically to form very long (0.5 to 3.0 cm) and thin (0.5 to 4 mm in diameter) multicellular structures . We describe the conditions required to obtain these stalk-like structures reproducibly in large numbers . Yeast mutants, mutated for control of cell polarity, developmental processes, UV response, and signal transduction cascades were tested and found capable of forming stalk-like structures . We suggest a model that explains the mechanism of stalk formation by mechanical environmental forces . We show that other microorganisms (Candida albicans, Schizosaccharomyces pombe, and Escherichia coli) also form stalks, suggesting that the ability to produce stalks may be a general property of microorganisms . Diploid yeast stalks sporulate at an elevated frequency, raising the possibility that the physiological role of stalks might be disseminating spores. Plant J, 1998 Apr, 14(1), 75 - 81 Molecular cloning and functional analysis of the Arabidopsis thaliana DNA ligase I homologue; Taylor RM et al.; A cDNA encoding the DNA ligase I homologue has been isolated from Arabidopsis thaliana using a degenerate PCR approach . The ORF of this cDNA encodes an amino acid sequence of 790 residues, representing a protein with a theoretical molecular mass of 87.8 kDa and an isoelectric point (pi) of 8.20 . Alignment of the A . thaliana DNA ligase protein sequence with the sequence of DNA ligases from human (Homo sapiens), murine (Mus musculus), clawed toad (Xenopus laevis) and the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae showed good sequence homology (42-45% identity, 61-66% similarity), particularly around the active site . Sequence data indicate that the Arabidopsis DNA ligase is the homologue of the animal DNA ligase I species . Functional analysis of the cDNA clone demonstrated its ability to complement the conditional lethal phenotype of an S . cerevisiae cdc9 mutant defective in DNA ligase activity, confirming that the cloned sequence encodes an active DNA ligase . The level of the DNA ligase transcript was not increased in A . thaliana seedlings in response to DNA damage induced by a period of enhanced UV-B irradiation . However, the cellular level of the DNA ligase mRNA transcript does correlate with the replicative state of plant cells. J Cell Biol, 1998 Jul 27, 142(2), 457 - 71 Regulation of cell polarity by microtubules in fission yeast; Sawin KE et al.; To investigate the role of microtubules in regulating cell polarity in Schizosaccharomyces pombe, we have developed a system in which normally cylindrical fission yeast synchronously form branched cells at high frequency upon treatment with the microtubule-depolymerizing drug thiabendazole (TBZ) . Branching depends on both elevated temperature and cell cycle state and occurs at high frequency only when TBZ is added to cells that have not yet passed through New-End Take-Off (NETO), the normal transition from monopolar to bipolar growth . This suggests that microtubules may be of greatest physiological importance for the maintenance of cell shape at specific points in the cell cycle . The localization of three different proteins normally found at cell ends-cortical F-actin, tea1, and an ral3 (scd2)-green fluorescent protein (GFP) fusion-is disrupted by TBZ treatment . However, these proteins can eventually return to cell ends in the absence of microtubules, indicating that although their localization to ends normally depends on microtubules, they may recover by alternative mechanisms . In addition, TBZ induces a shift in ral3-GFP distribution from cell ends to the cell middle, suggesting that a protein complex containing ral3 may be part of the cue that specifies the position of branch formation. J Biol Chem, 1998 Jul 31, 273(31), 19747 - 55 Characterization of the two small subunits of Saccharomyces cerevisiae DNA polymerase delta; Gerik KJ et al.; Yeast DNA polymerase delta (Poldelta) has three subunits of 125, 58, and 55 kDa . The gene for the 125-kDa catalytic subunit (POL3) has been known for several years . Here we describe the cloning of the genes for the 58- and 55-kDa subunits using peptide sequence analysis and searching of the yeast genome data base . The 58-kDa subunit, encoded by the POL31 gene, shows 23-28% sequence similarity to the 48-kDa subunit of human Poldelta and to S . pombe Cdc1 . POL31 is allelic to HYS2 and SDP5 . The 55-kDa subunit is encoded by the POL32 gene (ORF YJR043c in the yeast data base) . Very limited sequence similarity was observed between Pol32p and Schizosaccharomyces pombe Cdc27, the functionally analogous subunit in S . pombe Poldelta . The POL32 gene is not essential, but a deletion mutant shows cold sensitivity for growth and is sensitive to hydroxyurea and DNA damaging agents . In addition, lethality was observed when the POL32 deletion mutation was combined with conditional mutations in either the POL3 or POL31 gene . Pol32Delta strains are weak antimutators and are defective for damage-induced mutagenesis . The POL32 gene product binds proliferating cell nuclear antigen . A gel filtration analysis showed that Pol32p is a dimer in solution . When POL31 and POL32 were co-expressed in Escherichia coli, a tetrameric (Pol31p.Pol32p)2 species was detected by gel filtration, indicating that the two subunits form a complex. Yeast, 1998 Jun 15, 14(8), 747 - 57 Functional analysis of the Saccharomyces cerevisiae UBC11 gene; Townsley FM et al.; UBC11 is the Saccharomyces cerevisiae gene that is most similar in sequence to E2-C, a ubiquitin carrier protein required for the destruction of mitotic cyclins and proteins that maintain sister chromatid cohesion in animal cells and in Schizosaccharomyces pombe . We have disrupted the UBC11 gene and found it is not essential for yeast cell viability even when combined with deletion of UBC4, a gene that has also been implicated in mitotic cyclin destruction . Ubc11p does not ubiquitinate cyclin B in clam cell-free extracts in vitro and the destruction of Clb2p is not impaired in extracts prepared from delta ubc11 or delta ubc4 delta ubc11 cells . These results suggest Ubc4p and Ubc11p together are not essential for mitotic cyclin destruction in S . cerevisiae and we can find no evidence to suggest that Ubc11p is the true functional homologue of E2-C. Yeast, 1998 Jun 15, 14(8), 733 - 46 The C-terminal hydrophobic repeat of Schizosaccharomyces pombe heat shock factor is not required for heat-induced DNA-binding; Saltsman KA et al.; The C-terminal hydrophobic repeat (CTR) of heat shock transcription factor (HSF) has been proposed to regulate DNA binding by intramolecular interactions with the leucine zipper motifs present in the HSF trimerization domain . Schizosaccharomyces pombe provides a useful model organism for the study of the regulation of HSF DNA binding because, unlike Saccharomyces cerevisiae, S . pombe hsf is highly heat shock inducible for DNA binding and contains a clear homology to the CTR . We examined the role that the CTR plays in the regulation of S . pombe hsf by constructing isogenic strains bearing deletion and point mutations in the chromosomal copy of hsf . Surprisingly, we found that point mutation of key hydrophobic amino acids within the CTR, as well as full deletion of it, yielded factors that show normal binding at normal growth temperatures and full levels of heat-induced binding . Deletion of the CTR did, however, slightly lower the temperature required for maximal activation . In contrast, a large deletion of the C-terminus, which removes close to a third of the coding sequence, was deregulated and bound DNA at control temperature . Several of the deletion mutants were significantly reduced in their level of expression, yet they showed wild-type levels of DNA binding activity following heat shock . These experiments demonstrate that appropriate regulation of the DNA binding activity of S . pombe hsf is not solely dependent upon the CTR, and imply that a feedback mechanism exists that establishes proper levels of DNA binding following heat shock despite mutations that significantly alter levels of total hsf. Yeast, 1998 Jun 15, 14(8), 701 - 10 Pombe: a gene-finding and exon-intron structure prediction system for fission yeast; Chen T et al.; A special program developed by the authors, called Pombe, identifies protein coding regions in the Schizosaccharomyces pombe genome . Linear discriminant analysis was applied to predict 5'-terminal, internal, 3'-terminal exons (coding-exon) and introns . The accuracy of the prediction was tested by cross verifications . The sensitivity, specificity and correlation coefficient for the internal exon prediction were 98.5%, 99.9% and 98.3% respectively at the nucleotide level . Open reading frames were studied and used to predict intron-less genes: 99.0% of such genes were identified with correct stopping sites . The gene structure was determined by dynamic programming and the prediction achieved 97.0% correlation coefficient at the nucleotide level . The program is available at http:(/)/clio.cshl.org/genefinder. Mol Cell Biol, 1998 Aug, 18(8), 4488 - 98 An RNA binding protein negatively controlling differentiation in fission yeast; Tsukahara K et al.; The fission yeast Schizosaccharomyces pombe starts sexual development when starved for nutrients and simultaneously activated by mating pheromones . We have identified a new gene regulating the onset of this process . This gene, called nrd1(+), encodes a typical RNA binding protein that preferentially binds poly(U) . Deletion of nrd1(+) causes cells to initiate sexual development without nutrient starvation . We have found that the biological role of nrd1(+) is to block the onset of sexual development by repressing the Ste11-regulated genes essential for conjugation and meiosis until cells reach a critical level of starvation. Mol Gen Genet, 1998 Jun, 258(6), 663 - 70 Global control of meiotic recombination genes by Schizosaccharomyces pombe rec16 (rep1); Ding R et al.; The Schizosaccharomyces pombe rec16-125 mutation reduces meiotic recombination, delays premeiotic DNA synthesis, and reduces the accumulation of some but not other rec gene transcripts . To elucidate the function of the Rec16 global meiotic regulator, we cloned and sequenced rec16 . The data revealed that rec16 is identical to rep1, which was previously shown to encode a protein with a zinc-finger motif required for pre-meiotic DNA synthesis . Transcripts of rec16 (rep1) were strongly induced and subsequently degraded during meiosis . In a rec16 (rep1) deletion mutant, meiotic induction of the seven rec genes tested, which appear to be directly involved in meiotic recombination, was significantly reduced or essentially abolished . Deletion of 80% of the gene essentially abolished meiotic recombination, whereas strains deleted for approximately one-quarter of the gene, from either end, retained partial activity . The rec16-125 mutation strongly reduced recombination in the intervals tested on chromosomes I and III, a phenotype characteristic of mutations in rec genes, such as rec7, whose expression requires Rec16 (Rep1) . These results show that Rec16 (Rep1) does not have the regional specificity of Rec10 . We infer that Rec16 (Rep1) is a transcriptional activator that is required for meiotic replication and recombination because it plays a role in the transcriptional induction of the rec and other meiosis-specific genes. Biochem Cell Biol, 1998, 76(1), 107 - 13 Purification and kinetic characterization of hexokinase and glucose-6-phosphate dehydrogenase from Schizosaccharomyces pombe; Tsai CS et al.; Hexokinase and D-glucose-6-phosphate dehydrogenase (G6PDH) from Schizosaccharomyces pmbe have been purified 250-fold by an identical three-step . Both enzymes are dimeric with a molecular mass of 88 kDa for the kinase and 112 kDa for the dehydrogenase . Steady-state kinetic studies were performed on hexokinase and G6PDH, which form the glucose phosphate branch of the oxidative pentose phosphate pathway of S . pombe (fission yeast) . Hexokinase promotes Mg(2+)-activated phosphorylation of D-glucose by the equilibrium random Bi Bi mechanism with formation of the abortive enzyme-ADP-glucose complex . ADP inhibits the kinase competitively versus ATP and noncompetitively versus D-glucose . The Mg2+ activation of hexokinase is associated with an increase in the maximal velocity by its interaction with the ternary complex to facilitate the transfer of the phosphoryl group . G6PDH catalyzes NADP(+)-linked oxidation of D-glucose-6-phosphate by the ordered Bi Bi mechanism with NADP+ as the leading reactant . High NADP+ concentration inhibits the dehydrogenase by forming the dead-end ternary complex . In addition, G6PDH is also subjected to product inhibition by NADPH and noncompetitive inhibition by A(G)TP . Thus, the oxidative pentose phosphate pathway in S . pombe may be regulated via inhibition of hexokinase by ADP in conjunction with inhibition of G6PDH by NADPH and ATP. J Biol Chem, 1998 Jul 17, 273(29), 18490 - 8 Characterization of Pak2p, a pleckstrin homology domain-containing, p21-activated protein kinase from fission yeast; Sells MA et al.; p21-activated kinases (PAKs) bind to and are activated by Rho family GTPases such as Cdc42 and Rac . Since these GTPases play key roles in regulating cell polarity, stress responses, and cell cycle progression, the ability of PAK to affect these processes has been examined . We previously showed that fission yeast pak1+ encodes an essential protein that affects mating and cell polarity . Here, we characterize a second pak gene (pak2+) from Schizosaccharomyces pombe . Like the Saccharomyces cerevisiae proteins Cla4p and Skm1p, fission yeast Pak2p contains an N-terminal pleckstrin homology domain in addition to a p21-binding domain and a protein kinase domain that are common to other members of the PAK family . Unlike pak1+, pak2(+) is not essential for vegetative growth or for mating in S . pombe . Overexpression of the wild-type pak2+ allele suppresses the lethal growth defect associated with deletion of pak1+, and this suppression requires both the pleckstrin homology- and the p21-binding domains of Pak2p, as well as kinase activity . A substantial fraction of Pak2p is associated with membranous components, an association mediated both by the pleckstrin homology- and by the p21-binding domains . These results show that S . pombe encodes at least two pak genes with distinct functions and suggest that the membrane localization of Pak2p, directed by its interactions with membrane lipids and Cdc42p, is critical to its biological activity. J Biol Chem, 1998 Jul 17, 273(29), 18340 - 6 Identification of a human homologue of the Schizosaccharomyces pombe rad17+ checkpoint gene; Parker AE et al.; In the fission yeast Schizosaccharomyces pombe the rad17+ gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints . We have identified a human cDNA homologue of the S . pombe rad17+ checkpoint gene, designated Hrad17 . Hrad17 has 49% identity to the S . pombe rad17+ sequence at the DNA level and 49% identity and 72% similarity at the amino acid level . Northern blot analysis indicates elevated levels of expression in testis and in cancer cell lines . Chromosomal localization by fluorescence in situ hybridization indicates that Hrad17 is located on chromosome 4q13.3-21.2 . This region is subject to loss of heterozygosity in several human cancers . To begin to understand the protein-protein interactions of the human checkpoint machinery, we have used the yeast two-hybrid system to examine potential interactions between Hrad1, Hrad9, and Hrad17 . We demonstrate a physical interaction between Hrad17 and Hrad1 but no interaction with Hrad9. J Biol Chem, 1998 Jul 17, 273(29), 18332 - 9 A human homologue of the Schizosaccharomyces pombe rad1+ checkpoint gene encodes an exonuclease; Parker AE et al.; In the fission yeast Schizosaccharomyces pombe the rad1(+) gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints . We have identified a human homologue of the S . pombe rad1(+) gene, designated Hrad1, as well as a mouse homologue: Mrad1 . Two Hrad1 alternative splice variants with different open reading frames have been identified; one codes for a long form, Hrad1A, and the other encodes a short form because of N-terminal truncation, Hrad1B . Hrad1A has 60% identity to the S . pombe rad1+ sequence at the DNA level and 49% identity and 72% similarity at the amino acid level . Northern blot analysis indicates elevated levels of expression in testis and cancer cell lines . Chromosomal localization by fluorescence in situ hybridization indicates that Hrad1 is located on chromosome 5p13 . 2-13.3 . This region is subject to loss of heterozygosity in several human cancers . Hrad1 also shares homology with the Saccharomyces cerevisiae RAD17 and Ustilago maydis REC1 proteins . REC1 has previously been characterized as a 3' --> 5' exonuclease with a C-terminal domain essential for cell cycle checkpoint function . We have expressed and purified polyhistidine-tagged fusions of Hrad1A and Hrad1B and show that HisHrad1A has 3' --> 5' exonuclease activity, whereas HisHrad1B lacks such activity . The biological functions of the two proteins remain to be determined. Mol Pharmacol, 1998 Jul, 54(1), 213 - 9 Sensitivity to cisplatin and platinum-containing compounds of Schizosaccharomyces pombe rad mutants; Perego P et al.; The role of genes that affect response to radiation in determining sensitivity to platinum-containing compounds was studied using a panel of 23 strains of the yeast Schizosaccharomyces pombe . The radiation-hypersensitive mutants all had the same genetic background and most of them contained mutations that disabled either cell cycle checkpoints or DNA repair . The tested platinum compounds included cisplatin and two complexes containing diaminocyclohexane (oxaliplatin and tetraplatin), two ammine/cyclohexylamine complexes with different orientation of the leaving groups (JM216 and JM335) and a multinuclear platinum complex (BBR 3464) . The cytotoxic effect of the selected platinum complexes was evaluated by using a microtiter growth inhibition assay with a 48 hr exposure to drug . The mutants fell into three groups with respect to sensitivity to cisplatin: four mutants (rad2, -7, -11, -15) exhibited minimal change in sensitivity; fifteen mutants (rad4-6, -8-10, -12-14, -16-17, -19-21, and -22) were 5.1-21.7-fold hypersensitive; only rad1 and -3 mutants, defective in checkpoints, and rad18, defective in repair, displayed a marked hypersensitivity . None of the mutants demonstrated appreciable change in sensitivity to JM216 presumably as a consequence of a lack of resistance of the wild-type strain, whereas a moderate increase in sensitivity to JM335 was observed for most of the mutants, and hypersensitivity to BBR3464 was observed only in rad1 and -3 . No relevant changes in sensitivity to tetraplatin were observed . Most of the mutants, with the exception of rad2, -7, and -15, were hypersensitive to oxaliplatin . These findings demonstrate that specific mutations have disparate effects on the profile of sensitivity to different members of the same class of cytotoxic agents, which provides genetic evidence that different mechanisms are involved in differential cytotoxicity induced by Pt compounds . The results also demonstrate the utility of such a panel of mutants, constructed on the same genetic background, for detecting specific cellular response; presumably, this reflects the recognition or processing of specific DNA adducts . In conclusion, because the rad1 and rad3 gene products are determinants of cellular response to a large number of platinum-containing compounds, the present results support a critical role of genes involved in cell cycle control in cellular sensitivity to these agents. J Bacteriol, 1998 Jul, 180(14), 3727 - 9 A minisatellite sequence within the propeptide region of the vacuolar carboxypeptidase Y gene of Schizosaccharomyces pombe; Ingavale SS et al.; We describe the presence of a minisatellite sequence that displays length polymorphisms in the fission yeast Schizosaccharomyces pombe . The minisatellite sequence was found to reside within the propeptide region of the vacuolar carboxypeptidase Y gene . The minisatellite sequence, which was found only at a single locus, was mitotically stable and displayed length polymorphisms between the two varieties of S . pombe (S . pombe var . pombe and S . pombe var . malidevorans) . The minisatellite sequence, however, appeared to be species specific and was absent in other members of the Schizosaccharomyces genus . This report constitutes the first experimental demonstration of the presence of such sequences in yeasts. Mol Cell Biochem, 1998 Jun, 183(1-2), 125 - 32 Physiological consequences of expression of the Na+/H+ antiporter sod2 in Escherichia coli; Dibrov P et al.; Sod2 is the sodium-proton antiporter on the plasma membrane of the fission yeast Schizosaccharomyces pombe . It is vitally important for sodium export and pH homeostasis in this organism . Recently, the sod2 gene has been cloned and sequenced . However, initial attempts to express sod2 in Escherichia coli using the T7 promoter failed . In the present work we examined physiological consequences of expression of sod2 in E . coli . To alleviate problems caused by expression of sod2 we: (i) used sodium-free media at all steps; (ii) used the moderate tac promoter for expression and; (iii) used E . coli strain MH1 which has impaired sodium exchange . The effect of sod2 expression on E . coli varied depending on the E . coli genotype . When sod2 was expressed in BL21 cells which have normal Na+/H+ antiporters, the result was a Li+ sensitive phenotype . LiCl completely arrested or prevented growth of BL21 E . coli transformed with the sod2 gene . The effect on growth was pronounced in media of low external pH . Sod2 was then expressed in E . coli MH1 which is devoid of endogenous Na+/H+ antiporters . These cells became more resistant to external LiCl, but only in Na+ containing media . In the absence of external Na+, the presence of sod2 reduced growth . The results are explained in a model which demonstrates the physiological consequences of interference by expression of a foreign electroneutral Na+/H+ antiporter in conjunction with different housekeeping systems of E . coli host cells. Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 8159 - 64 sud1(+) targets cyclin-dependent kinase-phosphorylated Cdc18 and Rum1 proteins for degradation and stops unwanted diploidization in fission yeast; Jallepalli PV et al.; In the fission yeast Schizosaccharomyces pombe, S phase is limited to a single round per cell cycle through cyclin-dependent kinase phosphorylation of critical replication factors, including the Cdc18 replication initiator protein . Because defects in Cdc18 phosphorylation lead to a hyperstable and hyperactive form of Cdc18 that promotes high levels of overreplication in vivo, we wished to identify the components of the Cdc18 proteolysis pathway in fission yeast . In this paper we describe one such component, encoded by the sud1(+) gene . sud1(+) shares homology with the budding yeast CDC4 gene and is required to prevent spontaneous re-replication in fission yeast . Cells lacking sud1(+) accumulate high levels of Cdc18 and the CDK inhibitor Rum1, because they cannot degrade these two key cell cycle regulators . Through genetic analysis we show that hyperaccumulation of Rum1 contributes to re-replication in Deltasud1 cells, but is not the cause of the defect in Cdc18 proteolysis . Rather, Sud1 itself is associated with the ubiquitin pathway in fission yeast and binds to Cdc18 in vivo . Most importantly, Sud1-Cdc18 binding requires prior phosphorylation of the Cdc18 polypeptide at CDK consensus sites . These results provide a biochemical mechanism for the phosphorylation-dependent degradation of Cdc18 and other cell cycle regulators, including Rum1 . Evolutionary conservation of the Sud1/CDC4 pathway suggests that phosphorylation-coupled proteolysis may be a general feature of nearly all eukaryotic cell cycles. Gene, 1998 Jul 3, 214(1-2), 131 - 7 Identification of a novel casein kinase-1 homolog in fission yeast Schizosaccharomyces pombe; Kitamura K et al.; Fission yeast cells lacking either the ste9+- or rum1+ function cannot enter the cell differentiation pathway upon nutritional starvation . Sterility in both mutants is suppressed by the srs1-S41 mutation . A gene encoding a novel casein kinase-1 (CK1) isoform, cki3+, was isolated as a high-copy-number suppressor gene of the srs1 mutation . Cki3 protein is structurally more related to the Cki/Yck subfamily proteins than those of the Hhp/Hrr25 subfamily . A mutant cki3 gene in which a highly conserved lysine residue in the kinase subdomain II was substituted to arginine lost the ability to recover the growth defect in the srs1 mutant, indicating that catalytic activity was necessary for suppression . Gene disruption revealed that cki3+ was dispensable for cell viability, and cells lacking functional cki3+ exhibited no characteristic phenotype . Thus, S . pombe has three highly related CK1 isoforms (Cki1, Cki2 and Cki3), but none of them has an essential function. Gene, 1998 Jul 3, 214(1-2), 101 - 12 Mosaic structure of the cox2 gene in the petite negative yeast Schizosaccharomyces pombe: a group II intron is inserted at the same location as the otherwise unrelated group II introns in the mitochondria of higher plants; Schafer B et al.; In contrast to homologous genes in other fungal mitochondrial genomes, the gene encoding subunit 2 of cytochrome oxidase (cox2) in several Schizosaccharomyces pombe strains contains a large group II intron . Its 2436 nucleotides can be folded into a typical group II intron secondary structure, possessing all the expected sequence motifs for subgroup IIA1 (Michel et al., 1989) . This intron is remarkable for the following reasons: (i) Five nucleotide changes were observed compared with the continuous form of the cox2 gene in the reference strain 50 at the 3'-exon sequence, but not in the 5'-exon . (ii) One of these changes occurred at the splice point leading to a serine instead of a threonine residue in the deduced cox2 polypeptide . In all cases, the alterations resulted in the replacement of more frequently used codons by rare ones . (iii) Although the intron is able to undergo splicing, the sequence motifs thought to be necessary for interaction between the 5'-exon and the intron during the splicing process (the EBS1/IBS1 as well as the EBS2/IBS2 pairings) are unusual . (iv) The intron is inserted at the same location in the cox2 gene as the otherwise unrelated intron from higher plants. Nucleic Acids Res, 1998 Jul 15, 26(14), 3319 - 22 Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases; Gross T et al.; We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins . All contain the Homol D-box in their promoter . We have shown that Homol D is, in this promoter type, the TATA-analogue . Many promoters contain the Homol E-box, which serves as a proximal activation sequence . Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28 . The budding yeast Saccharomyces cerevisiae has no L28 equivalent . Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes . Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast. Genetics, 1998 Jul, 149(3), 1265 - 75 Isolation and characterization of new fission yeast cytokinesis mutants; Balasubramanian MK et al.; Schizosaccharomyces pombe is an excellent organism in which to study cytokinesis as it divides by medial fission using an F-actin contractile ring . To enhance our understanding of the cell division process, a large genetic screen was carried out in which 17 genetic loci essential for cytokinesis were identified, 5 of which are novel . Mutants identifying three genes, rng3(+), rng4(+), and rng5(+), were defective in organizing an actin contractile ring . Four mutants defective in septum deposition, septum initiation defective (sid)1, sid2, sid3, and sid4, were also identified and characterized . Genetic analyses revealed that the sid mutants display strong negative interactions with the previously described septation mutants cdc7-24, cdc11-123, and cdc14-118 . The rng5(+), sid2(+), and sid3(+) genes were cloned and shown to encode Myo2p (a myosin heavy chain), a protein kinase related to budding yeast Dbf2p, and Spg1p, a GTP binding protein that is a member of the ras superfamily of GTPases, respectively . The ability of Spg1p to promote septum formation from any point in the cell cycle depends on the activity of Sid4p . In addition, we have characterized a phenotype that has not been described previously in cytokinesis mutants, namely the failure to reorganize actin patches to the medial region of the cell in preparation for septum formation. Genetics, 1998 Jul, 149(3), 1251 - 64 A screen for genes involved in the anaphase proteolytic pathway identifies tsm1(+), a novel Schizosaccharomyces pombe gene important for microtubule integrity; Grishchuk EL et al.; The growth of several mitotic mutants of Schizosaccharomyces pombe, including nuc2-663, is inhibited by the protease inhibitor N-Tosyl-L-Phenylalanine Chloromethyl Ketone (TPCK) . Because nuc2(+) encodes a presumptive component of the Anaphase Promoting Complex, which is required for the ubiquitin-dependent proteolysis of certain proteins during exit from mitosis, we have used sensitivity to TPCK as a criterion by which to search for novel S . pombe mutants defective in the anaphase-promoting pathway . In a genetic screen for temperature-sensitive mitotic mutants that were also sensitive to TPCK at a permissive temperature, we isolated three tsm (TPCK-sensitive mitotic) strains . Two of these are alleles of cut1(+), but tsm1-512 maps to a novel genetic location . The tsm1-512 mutation leads to delayed nuclear division at restrictive temperatures, apparently as a result of an impaired ability to form a metaphase spindle . After shift of early G2 cells to 36 degrees, tsm1-512 arrests transiently in the second mitotic division and then exits mitosis, as judged by spindle elongation and septation . The chromosomes, however, often fail to segregate properly . Genetic interactions between tsm1-512 and components of the anaphase proteolytic pathway suggest a functional involvement of the Tsm1 protein in this pathway. Genetics, 1998 Jul, 149(3), 1221 - 33 Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity; Gould KL et al.; A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast . We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1 . The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD . A cdc24 null mutant was generated by homologous recombination . Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene . The transcript of cdc24(+) is present at constant levels throughout the cell cycle . Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase . Arrest is accompanied by a rapid loss of viability and chromosome breakage . An S . pombe homolog of the replicative DNA helicase DNA2 of S . cerevisiae suppresses cdc24 . These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity. Genetics, 1998 Jul, 149(3), 1191 - 204 The M26 hotspot of Schizosaccharomyces pombe stimulates meiotic ectopic recombination and chromosomal rearrangements; Virgin JB et al.; Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination) . Recombination hotspots are important elements in controlling meiotic allelic recombination . We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination . Ectopic recombination was reduced 10-1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S . pombe . The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination . Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35-60% of recombination events and was stimulated 12-fold by M26 . These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements. Genes Dev, 1998 Jul 1, 12(13), 1986 - 97 Identification of Xenopus SMC protein complexes required for sister chromatid cohesion; Losada A et al.; The structural maintenance of chromosomes (SMC) family is a growing family of chromosomal ATPases . The founding class of SMC protein complexes, condensins, plays a central role in mitotic chromosome condensation . We report here a new class of SMC protein complexes containing XSMC1 and XSMC3, Xenopus homologs of yeast Smc1p and Smc3p, respectively . The protein complexes (termed cohesins) exist as two major forms with sedimentation coefficients of 9S and 14S . 9S cohesin is a heterodimer of XSMC1 and XSMC3, whereas 14S cohesin contains three additional subunits . One of them has been identified as a Xenopus homolog of the Schizosaccharomyces pombe Rad21p implicated in DNA repair and the Saccharomyces cerevisiae Scc1p/Mcd1p implicated in sister chromatid cohesion . 14S cohesin binds to interphase chromatin independently of DNA replication and dissociates from it at the onset of mitosis . Immunodepletion of cohesins during interphase causes defects in sister chromatid cohesion in subsequent mitosis, whereas condensation is unaffected . These results suggest that proper assembly of mitotic chromosomes is regulated by two distinct classes of SMC protein complexes, cohesins and condensins. Mol Gen Genet, 1998 May, 258(4), 389 - 96 Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe; Neu R et al.; In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomzyces pombe . In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome . Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus . As host strain for the ectopic expression, we used the nonsense mutant ana(r)-6, which carries a premature stop codon in the urf a gene . The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol . The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene . Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae . We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana(r)-6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype . The significant similarity of the urf a gene to the mitochondrial var1 gene of S . cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity. Mol Gen Genet, 1998 May, 258(3), 279 - 87 Alteration of the largest subunit of RNA polymerase II and its effect on chromosome stability in Schizosaccharomyces pombe; Sugaya K et al.; A mutation in the RNA polymerase II largest subunit (RpII LS) that is related to abnormal induction of sister chromatid exchange has previously been described the CHO-K1 cell mutant tsTM4 . To elucidate the molecular basis of this effect we introduced the mutation into the homologous site in the Schizosaccharomyces pombe rpbl gene, which encodes RpII LS . Since the tsTM4 mutant exhibited a decrease in the rate of DNA synthesis in cells arrested in S phase at the nonpermissive temperature, we focussed on the study of growth, the cell cycle, and chromosome stability at various temperatures . First, we examined the effects of the mutation on haploid yeast cells . The mutant showed slower growth than the wild type, but cell growth was not arrested at the nonpermissive temperature . When growing cells were shifted to the nonpermissive temperature, an accumulation of cells in G1 and/or G0 was observed . Tetrad analysis suggested that these phenotypes were associated with the mutation . In diploid cells, chromosome instability was detected by loss of intragenic complementation between two alleles of the ade6 gene . An abnormal fraction of cells containing an intermediate DNA content was also observed by FACS analysis . The accumulation of this fraction may reflect the fact that a large number of cells are in S phase or have an abnormal DNA content as a result of chromosome instability . These observations demonstrate that the S . pombe rpb1 mutant exhibits a phenotype very similar to that of the CHO-K1 cell mutant tsTM4. Curr Genet, 1998 Jun, 33(6), 445 - 50 Isolation of a Trichoderma reesei cDNA encoding GTP: a-D-mannose-1-phosphate guanyltransferase involved in early steps of protein glycosylation; Kruszewska JS et al.; A cDNA coding for GTP: alpha-d-mannose-1-phosphate guanyltransferase (MPG1 transferase) (EC 2.7.7.13) was isolated from a cDNA library of the Trichoderma reesei RutC-30 strain by suppression of the yeast Saccharomyces cerevisiae mutation in the DPM1gene encoding mannosylphosphodolichol (MPD) synthase . The nucleotide sequence of the 1.6 kb-long cDNA revealed an ORF which encodes a protein of 364 amino acids . Sequence comparisons demonstrate 70% identity with the S . cerevisiae guanyl transferase gene (MPG1) and 75% identity with the Schizosaccharomyces pombe homologue . No similarity was found with the MPD synthase encoded by the S . cerevisiae DPM1 gene . The possibility that cloned cDNA encodes a product with a MPD synthase activity was also excluded by transforming a heterozygous S . cerevisiae dpm1::LEU2/DPM1 diploid, which did not lead to the restoration of viability of the dpm1 spores . Simultaneously, a significant increase in MPG transferase activity, as compared with the wild-type yeast, was observed in cellular extracts when the mpg1 cDNA from Trichoderma was expressed in the S . cerevisiae dpm1-6 mutant. Mol Microbiol, 1998 May, 28(4), 729 - 41 Schizosaccharomyces pombe isp4 encodes a transporter representing a novel family of oligopeptide transporters; Lubkowitz MA et al.; We have recently cloned an oligopeptide transport gene from Candida albicans denoted OPT1 . This gene showed significant sequence similarity to three open reading frames (ORFs) with no previously established function: isp4 from Schizosaccharomyces pombe and Saccharomyces cerevisiae YJL212C and YPR194C, identified during the genome project . The S . pombe gene isp4 was originally identified by Sato et al . as a gene that was upregulated through nitrogen starvation induction of meiosis . However, an isp4delta strain exhibited a wild-type phenotype with respect to sexual differentiation . We have found that the same isp4delta strain is deficient in tetrapeptide transport activity as measured by its resistance to toxic tetrapeptides, by its inability to accumulate a radiolabelled tetrapeptide and by the inability to use tetrapeptides as a sole source of an amino acid to satisfy an auxotrophic requirement . Similarly, we found that the ORF YPR194C from S . cerevisiae encodes an oligopeptide transporter . Sequence analyses as well as physiological evidence has led us to propose that the proteins encoded by isp4 and the genes identified from S . cerevisiae and C . albicans comprise a new group of transporters specific for small oligopeptides, which we have named the OPT family. J Mol Biol, 1998 Jun 19, 279(4), 703 - 12 Two large subunits of the fission yeast RNA polymerase II provide platforms for the assembly of small subunits; Ishiguro A et al.; The subunit-subunit contact network was analyzed for the Schizosaccharomyces pombe RNA polymerase II consisting of ten putative subunits . Previously we carried out far-Western blot analysis of bimolecular interaction with radio-labeled subunit 3 and 5 probes . Here we extended the analysis using another six small-sized subunits as probes . Taking the results together the subunit-subunit interaction was observed for a total 18 (or 19) combinations . All eight small-sized subunits exhibited binding activities to two large subunits, Rpb1 and Rpb2 . In addition, bimolecular interaction was observed for the combinations of Rpb3-Rpb5, Rpb3-Rpb11 (and Rpb5-Rpb8/11) . The subunit-subunit contact within the assembled RNA polymerase was then analyzed by protein-protein cross-linking using five species of bifunctional cross-linkers with different length and specificity . Cross-linking was observed for a total of 19 combinations, including five combinations between small subunits, Rpb3-Rpb10, Rpb3-Rpb11, Rpb5-Rpb6, Rpb6-Rpb7 and Rpb6-Rpb8 . The results altogether indicate that two large subunits Rpb1 and Rpb2 provide the platform for assembly of small subunits and also small subunits interact with each other for limited combinations . Direct contact of the two large subunits, Rpb1 and Rpb2, was also demonstrated by cross-linking . EMBO J, 1998 Jun 1, 17(11), 3066 - 77 Nascent transcription from the nmt1 and nmt2 genes of Schizosaccharomyces pombe overlaps neighbouring genes; Hansen K et al.; We have determined the extent of the primary transcription unit for the two highly expressed genes nmt1 and nmt2 of Schizosaccharomyces pombe . Transcription run-on analysis in permeabilized yeast cells was employed to map polymerase density across the 3'-flanking region of these two genes . Surprisingly, polymerases were detected 4.3 kb beyond the nmt1 polyadenylation {poly(A)} site and 2.4 kb beyond the nmt2 poly(A) site, which in each case have transcribed through an entire convergent downstream transcription unit . However, the steady-state levels of both downstream genes were unaffected by the high level of nmt1 or nmt2 nascent transcription . Analysis of nmt1 and nmt2 RNA 3' end formation signals indicates that efficient termination of transcription requires not only a poly(A) signal but also additional pause elements . The absence of such pause elements close to the poly(A) sites of these genes may account for their extended nascent transcripts. Mol Cell Biol, 1998 Jul, 18(7), 4141 - 8 Functional conservation of the transportin nuclear import pathway in divergent organisms; Siomi MC et al.; Human transportin1 (hTRN1) is the nuclear import receptor for a group of pre-mRNA/mRNA-binding proteins (heterogeneous nuclear ribonucleoproteins {hnRNP}) represented by hnRNP A1, which shuttle continuously between the nucleus and the cytoplasm . hTRN1 interacts with the M9 region of hnRNP A1, a 38-amino-acid domain rich in Gly, Ser, and Asn, and mediates the nuclear import of M9-bearing proteins in vitro . Saccharomyces cerevisiae transportin (yTRN; also known as YBR017c or Kap104p) has been identified and cloned . To understanding the nuclear import mediated by yTRN, we searched with a yeast two-hybrid system for proteins that interact with it . In an exhaustive screen of the S . cerevisiae genome, the most frequently selected open reading frame was the nuclear mRNA-binding protein, Nab2p . We delineated a ca.-50-amino-acid region in Nab2p, termed NAB35, which specifically binds yTRN and is similar to the M9 motif . NAB35 also interacts with hTRN1 and functions as a nuclear localization signal in mammalian cells . Interestingly, yTRN can also mediate the import of NAB35-bearing proteins into mammalian nuclei in vitro . We also report on additional substrates for TRN as well as sequences of Drosophila melanogaster, Xenopus laevis, and Schizosaccharomyces pombe TRNs . Together, these findings demonstrate that both the M9 signal and the nuclear import machinery utilized by the transportin pathway are conserved in evolution. Mol Cell Biol, 1998 Jul, 18(7), 4097 - 108 Myb-related Schizosaccharomyces pombe cdc5p is structurally and functionally conserved in eukaryotes; Ohi R et al.; Schizosaccharomyces pombe cdc5p is a Myb-related protein that is essential for G2/M progression . To explore the structural and functional conservation of Cdc5 throughout evolution, we isolated Cdc5-related genes and cDNAs from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens . Supporting the notion that these Cdc5 gene family members are functionally homologous to S . pombe cdc5(+), human and fly Cdc5 cDNAs are capable of complementing the temperature-sensitive lethality of the S . pombe cdc5-120 mutant . Furthermore, S . cerevisiae CEF1 (S . cerevisiae homolog of cdc5(+)), like S . pombe cdc5(+), is essential during G2/M . The location of the cdc5-120 mutation, as well as mutational analyses of Cef1p, indicate that the Myb repeats of cdc5p and Cef1p are important for their function in vivo . However, we found that unlike in c-Myb, single residue substitutions of glycines for hydrophobic residues within the Myb repeats of Cef1p, which are essential for maintaining structure of the Myb domain, did not impair Cef1p function in vivo . Rather, multiple W-to-G substitutions were required to inactivate Cef1p, and many of the substitution mutants were found to confer temperature sensitivity . Although it is possible that Cef1p acts as a transcriptional activator, we have demonstrated that Cef1p is not involved in transcriptional activation of a class of G2/M-regulated genes typified by SWI5 . Collectively, these results suggest that Cdc5 family members participate in a novel pathway to regulate G2/M progression. Mol Cell Biol, 1998 Jul, 18(7), 3782 - 7 Tyrosine phosphorylation of cdc2 is required for the replication checkpoint in Schizosaccharomyces pombe; Rhind N et al.; The DNA replication checkpoint inhibits mitosis in cells that are unable to replicate their DNA, as when nucleotide biosynthesis is inhibited by hydroxyurea . In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that this checkpoint involves the inhibition of Cdc2 activity through the phosphorylation of tyrosine-15 . On the contrary, a recent biochemical study indicated that Cdc2 is in an activated state during a replication checkpoint, suggesting that phosphorylation of Cdc2 on tyrosine-15 is not part of the replication checkpoint mechanism . We have undertaken biochemical and genetic studies to resolve this controversy . We report that the DNA replication checkpoint in S . pombe is abrogated in cells that carry the allele cdc2-Y15F, expressing an unphosphorylatable form of Cdc2 . Furthermore, Cdc2 isolated from replication checkpoint-arrested cells can be activated in vitro by Cdc25, the tyrosine phosphatase responsible for dephosphorylating Cdc2 in vivo, to the same extent as Cdc2 isolated from cdc25ts-blocked cells, indicating that hydroxyurea treatment causes Cdc2 activity to be maintained at a low level that is insufficient to induce mitosis . These studies show that inhibitory tyrosine-15 phosphorylation of Cdc2 is essential for the DNA replication checkpoint and suggests that Cdc25, and/or one or both of Wee1 and Mik1, the tyrosine kinases that phosphorylate Cdc2, are regulated by the replication checkpoint. Curr Biol, 1998 May 21, 8(11), 633 - 41 Cut1 is loaded onto the spindle by binding to Cut2 and promotes anaphase spindle movement upon Cut2 proteolysis; Kumada K et al.; BACKGROUND: The Cut1 and Cut2 proteins of the fission yeast Schizosaccharomyces pombe form a complex and are required for the separation of sister chromatids during anaphase . Polyubiquitinated Cut2 degrades at the onset of anaphase and this degradation, like that of mitotic cyclin, is dependent on the anaphase-promoting complex/cyclosome . Expression of Cut2 that cannot be degraded blocks sister chromatid separation and anaphase spindle elongation . Here, we have investigated the role of the Cut1-Cut2 interaction in sister chromatid separation . RESULTS: The carboxyl terminus of Cut2 interacts with the amino terminus of Cut1, and temperature-sensitive Cut2 mutants expressed Cut2 proteins that contain substitutions in the carboxyl terminus and fail to interact with Cut1, resulting in aberrant anaphase . Localization of Cut1 alters dramatically during the cell cycle . Cut1 is retained in the cytoplasm during interphase and moves to the mitotic spindle pole bodies and the spindle upon entry into prophase, when spindles are formed . The association between Cut2 and Cut1 is needed for the localization of Cut1 to the spindles, as Cut1 remains unbound to the spindle if complex formation is impaired . Cut2 degrades during anaphase, but Cut1 remains bound to the anaphase spindle . This association with the anaphase spindle requires the conserved carboxyl terminus of Cut1 . CONCLUSIONS: Complex formation between Cut1 and Cut2 is needed for the onset of normal anaphase . Cut2 is required for loading Cut1 onto the spindle at prophase and Cut2 proteolysis is needed for the active participation of Cut1 in sister chromatid separation. Curr Biol, 1998 May 21, 8(11), 611 - 21 Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body; Eng K et al.; BACKGROUND: An actomyosin-based contractile ring plays a pivotal role in cytokinesis . Despite the identification of many components of the ring, the steps involved in its assembly are unknown . The fission yeast Schizosaccharomyces pombe is an attractive organism in which to study cytokinesis because its cell cycle has been well characterized; it divides by medial fission using an actomyosin ring; and a number of S . pombe mutants defective in actomyosin ring assembly have been isolated . Here, we have characterized one such mutant, rng2 . RESULTS: Temperature-sensitive rng2 mutants accumulated F-actin cables in the medial region of the cell but failed to organize the cables into a ring . In rng2-null mutants, only a spot-like structure containing F-actin was detected . The rng2+ gene encodes a protein related to human IQGAP1, a protein that binds actin and calmodulin and is a potential effector for the Rho family of GTPases . Rng2p localized to the actomyosin ring and to the spindle pole body (SPB) of interphase and mitotic cells . Localization of Rng2p to the actomyosin ring but not the SPB required F-actin . Rng2p interacted with calmodulin, a component of the SPB and the actomyosin ring . The rng2 gene showed genetic interactions with three other actomyosin ring assembly mutants, cdc4, cdc12, and rng5 . CONCLUSIONS: The S . pombe IQGAP-related protein Rng2p is a component of the actomyosin ring and the SPB and is required for actomyosin ring construction following assembly of F-actin at the division site. Yakugaku Zasshi, 1998 Apr, 118(4), 112 - 26 Natural organic compounds that affect to microtubule functions; Iwasaki S; Microtubules (MT), composed of a protein tubulin (TN) alpha,beta-heterodimer with concomitant other proteins, microtubule associated proteins (MAPs and tau), are known to be the main component of spindles in a mitotic apparatus of eucaryotic cells, and are also involved in many other basic and essential cell functions . There are a number of natural and synthetic compounds that interfere with MT function to cause the mitotic arrest of eucaryotic cells . Such antimitotic agents show a broad biological activity, and can be used for medicinal and agrochemical purposes . On the other hand, they are also important as the biochemical tools for understanding the dynamics of MT network . Most of such antimitotic agents, with a few exceptions, bind to beta-TN . Among them, colchicine (CLC), vinblastine (VLB) and taxol have been of major importance in biochemical studies of MT and in studies of their intracellular functions . The former two both inhibit MT assembly but their binding sites on beta-TN are different; CLC-site and VLB-site, and many MT inhibitors bind to either sites . Taxol bind to TN at a site other than CLC-site and VLB-site, and promote MT assembly . We have worked on a variety of antimitotic agents that bind to CLC, VLB or taxol-site, in discoveries, structures, biological actions and/or interactions with TN . In this paper, I summarized the results of our studies on VLB-site ligands; (1) rhizoxin (RZX), isolated as a phytotoxin produced by a plant pathogenic fungus, and its related compounds, (2) derivatives of ansamitocin P-3 (ASMP3) (maytansinoid: MAY), isolated as a cytotoxic metabolite of an Actinomycete, (3) phomopsin A (PMSA), isolated as a mycotoxin produced by a plant parasitic fungus, (4) dolastatin 10 (DLS10), isolated as a cytotoxic metabolite of a see animal, (5) ustiloxins (USL) A-F, isolated as a mycotoxin produced by a plant pathogenic fungus, (6) arenastation A (ARSA), isolated as a cytotoxic metabolite of a sponge, and its synthetic analogs . From our studies on interactions of these VLB-site ligands with TN, we showed that the presence of a distinct RZX/MAY-binding site which only partially overlap with VLB-site, and that PMSA, DLS10, USLs and ARSA bind to the RZX/MAY site . RZX, ASMP3 and ARSA inhibit the growth of a variety of fungi, including Aspergillus nidulans . In order to obtain information as to the drug-TN interaction at the RZX/MAY site, RZX-resistant beta-TN gene mutants were isolated from RZX-sensitive wild-type A . nidulans . In all the beta-TN gene mutants, single amino acid (100th) alteration, asparagine-to-isoleucine, was observed . Sequence displacement experiments confirmed that this alteration conferred resistance to RZX and ASMP3, and also to ARSA . This resistance mechanism was further verified with yeasts Schizosaccharomyces pombe and Saccharomyces serevisiae . All the natural ligands mentioned above show potent cytotoxicity against human and murine tumor cells, but VLB, PMSA, DLS10 and USLA are inactive to both RZX-sensitive and -resistant fungal strains. Nat Biotechnol, 1996 May, 14(5), 600 - 5 A functional screen in yeast for regulators and antagonizers of heterologous protein tyrosine kinases; Superti-Furga G et al.; Tyrosine phosphorylation exerts a pivotal r